Viruses causing Cancer (HPV) Mechanisms of Human Disease 20190416 at 11:00 am TM Gallagher

COMMON FEATURES OF THE CANCER-CAUSING VIRUSES Viruses are persistently infecting, lasting years in patients Viruses are geographically and culturally-restricted (“infectious” cancers) Viruses are more common than the tumors they cause (infection alone is not sufficient to cause cancer; infection is one “hit” in “multi-hit” carcinogenesis) Viruses operate “directly”, in that the virus genomes , or parts of the genomes, are in the tumor cells (this does not apply, however, to HIV and HCV)

FEMALES Virus-associated cancers: ~ 10-20% of cancers are associated with virus infections. Cervical cancers (blue) are the most common virus-associated cancers in females. Liver cancers (purple) are the most common virus-associated cancers in males. MALES

How do viruses cause cancers? Papillomaviruses can be used as an example to (partially) answer this question. Papillomaviruses cause warts (a form of epithelial dysplasia). Some papillomavirus types can cause more severe epithelial carcinomas.

HPVs can be collected from warts: From each type of wart (plantar warts, flat warts, anal or cervical warts, etc.), a distinct “type” of HPV is isolated. There are nearly 100 HPV types Type classification is based on relatedness of the HPV DNA genomes Common warts

HPV types are divided into cutaneous and mucosal groups Note that each HPV type typically presents a distinct clinical picture, i.e., a cutaneous wart such as HPV1 will not cause a mucosal disease such as anogenital condyloma.

Cutaneous HPVs cause warts with distinct morphologies Types 2, 4 Types 3, 10 Types 1, 2, 4 Types 5, 8, 17, 20, 36

HPVs in the mucosal group are divided into high- and low-risk categories

High-risk mucosal HPVs cause cervical intraepithelial neoplasias (CIN), which can advance to cervical carcinomas

HPVs are nonenveloped DNA viruses (very stable viruses) that infect through breaks in epithelial layers HPV

The infection process that produces new HPV particles is tied to epithelial cell differentiation

Epithelial cell differentiation is correlated with HPV gene expression The E “early” genes are transcribed shortly after basal keratinocytes are infected. The L “late” gene products are transcribed in the terminally-differentiated upper cell layers.

Several months elapse between inoculation and appearance of wart(s)

How does HPV cause epithelial cancers? Cell transformation comes from abortive events that take place rarely and “stochastically” after infection of the basal cells

HPV integration into the host genome: An abortive event E2 gene is “broken” E6 and E7 genes are intact (note arrows) Constitutive E6 and E7 production from integrated HPV DNA

p53 Rb

Viruses thwart this response to maintain themselves in nature. p53 and Rb proteins can be viewed as part of an innate antiviral defense mechanism. p53 increases in response to some DNA virus infections, thereby causing cell (and thus virus) death. Viruses thwart this response to maintain themselves in nature. Some viruses interfere more effectively than others; some viruses are more tumorigenic than others. p53 Rb

Why are HPVs 16 and 18 “high – risk”, while many others are “low-risk” for cancer?

Cervical carcinomas can be invasive (spread to regional lymph nodes) DIAGNOSIS: IN HPV NEOPLASIA : LOOK FOR DYSPLASTIC CELLS Cervical carcinomas can be invasive (spread to regional lymph nodes) but metastasis beyond the pelvis is rare (only seen in very advanced disease)

DIAGNOSIS: “Koilocytic” cells are hallmarks of HPV-induced dysplasia DNA probe for HPV (“in situ” hybridization): Note nuclei stain positive for HPV DNA. Nuclei are surrounded by vacuoles. Papinocolau stain: Note vacuolated cells and haloes around nuclei ~ 5% of all Pap smears contain HPV-infected cells Development of koilocytic cells develops years after HPV infection

DIAGNOSIS: RISH: RNA In Situ Hybridization; note HPV E6 and E7 in dysplastic epithelia In situ hybridization can identify HPV even when there is no evidence of dysplasia (i.e., neg. on pap smear)

THERAPY: Indications to remove warts: Pain, cosmetic reasons, or in the case of laryngeal papillomas, remove airway obstructions Treatments: - Surgical cryotherapy or electro cautery - Podophyllotoxin cream [podophyllotoxin; aka PPT; is a plant-derived toxin that damages DNA to kill HPV-infected cells] - Imiquimod cream [imiquimod is a synthetic TLR7 agonist that induces proinflammatory cytokines, notably interferons, which attract antiviral effector cells to destroy warts] - Cidofovir [cidofovir is a nucleoside analog that blocks DNA polymerases; standard use is to reduce herpes infections but topical cidofovir has been shown to reduce some HPV-induced warts]

VACCINATION: GARDASIL Contains virus-like particles (VLPs) made in yeast cells (vaccine is not recommended for patients with yeast allergies) Vaccine contains four HPV VLPs (types 6, 11, 16, 18). Vaccine can offer cross-protection to some other HPV type, i.e., HPV58 Given in three injections Protects against ~ 70% of cervical cancer (other HPV types not present in the vaccine account for the remaining ~ 30% of cancers) Ineffective if patient already has CIN

Note pharyngeal HPV-associated cancers in males HEAD AND NECK CANCERS are an under – appreciated justification for HPV vaccination for both girls and boys. HPV (typically HPV 16, which is included in the vaccine) can infect the squamous epithelia of the nasopharynx and oropharynx. HPV E6 and E7 contribute to development of squamous cell carcinomas of the the head and neck (same MOA as for HPV-induced developments of cervical carcinomas). Smoking, alcohol abuse and obesity are additional risk factors for developing head and neck cancers

Chlamydia trachomatis Herpes simplex virus 1 Human papillomavirus A 31-year-old woman presents to her gynecologist for a routine well-visit. She is sexually active with multiple male partners and uses an intrauterine device for contraception. Her last menstrual period was two weeks ago. She denies abnormal vaginal discharge or sensations of burning or itching. Pelvic exam is normal. Routine Pap smear shows the following (see Image A). Which organism is most likely responsible for her abnormal Pap smear? Treponema pallidum Chlamydia trachomatis Herpes simplex virus 1 Human papillomavirus Trichomonas vaginalis DISCUSSION: The Pap smear in Image A shows koilocytes, which are cervical epithelial cells with structural changes associated with human papillomavirus (HPV) infection. Koilocytes are atypical epithelial cells that can be seen on biopsy or Pap smear and are suggestive of human papillomavirus (HPV) infection. Koilocytes are identified by their increased nuclear to cytoplasmic ratio, darkening of the nucleus or hyperchromasia, and clearing of the area surrounding the nucleus. As HPV infection is associated with increased cervical cancer risk, the presence of koilocytes on a Pap smear is suspicious for cervical cancer or a pre-malignant lesion. Apgar and Brotzman discuss the role of Pap smears in the diagnosis and evaluation of HPV infection and associated cervical cancer. The presence of minimal atypia, called "atypical squamous cells of undetermined significance" (ASC-US), is an indication for HPV testing. Specifically, HPV typing for high-risk vs low-risk oncogenic types is important for determining risk for progression to malignancy. Krawczyk et al. describe how HPV infection leads to koilocyte development. They were able to demonstrate how specific proteins found in both low-risk and high-risk HPV types act together to induce koilocyte formation. Specifically, these proteins are responsible for the peri-nuclear clearing that is characteristic of koilocytic cells. Image A shows a Pap smear with koilocytes present on the right and normal squamous epithelial cells on the top and left. Illustration A shows an alternative appearance of koilocytes on Pap smear, with normal squamous epithelial cells on the left and koilocytes on the right. Incorrect Answers: Answer 1: Treponema pallidum causes syphilis. Syphilis is diagnosed via direct visualization of spirochetes with dark-field microscopy or fluorescent antibodies, or via serum antibody screening with VDRL or FTA-ABS tests. Answer 2: Infection with chlamydia trachomatis can be diagnosed by visualization of cytoplasmic inclusions using Giemsa stain or fluorescent antibody stain. Answer 3: Infection with Herpes simplex virus 1 is often diagnosed by its classic appearance, but confirmation is assisted by Tzank smear, polymerase chain reaction or culture of an opened skin vesicle. Answer 5: Trichomonas vaginalis causes trichomonas vaginitis. Motile trophozoites can be directly visualized via light microscopy in vaginal discharge.